PROJECT SUMMARY Analytic treatment interruption (ATI) studies remain the ultimate test of efficacy for HIV curative strategies, which will need to demonstrate a significant delay of viral rebound for clinical approval. ATI trials will become increasingly important as new HIV curative strategies are discovered and advance in clinical trials. However, there is a current knowledge gap in our understanding of factors that affect the timing of viral rebound and the sources of rebounding virus. This has led to significant challenges in designing novel therapeutics for HIV remission and evaluating their efficacy in pre-clinical and early phase clinical trials. In this application, we propose a collaborative effort by a multidisciplinary team of virologists, immunologists, and mathematicians to tackle these challenges by studying a rare cohort of participants from prior AIDS Clinical Trials Group ATI trials. We plan to define virologic characteristics associated with viral rebound timing and rebounding viral variants. This includes assessing the impact of immune escape mutations, quantifying viral RNA species within cellular subsets, and determining the integration site characteristics of rebounding virus. Concurrently, we plan to perform an in-depth evaluation of both cellular, antibody, and inflammatory markers associated with HIV rebound timing. Finally, we will use the results of this study to enhance a mathematical model of post- treatment HIV control. The knowledge gained will inform the design and evaluation of novel strategies for drug- free HIV remission.